Method for the determination of pseudomonas in biological media

ABSTRACT

The presence of Pseudomonas in biological media containing other micro-organisms is determined by adding to the media from 100 to about 800 parts per million of a compound of the formula:   WHEREIN R is halogen and R1 is hydrogen or chloro. The medium is then maintained at 20* to 40* C. for an incubation period of 12 hours to 14 days. A particularly preferred compound useful in this method is 2,4,4&#39;&#39;-trichloro-2&#39;&#39;-hydroxydiphenyl ether.

United States Patent [72] Inventor Thomas E. Furia Hartsdale, N.Y. [21] Appl. No. 714,089 [22] Filed Mar. 18, 1968 [45] Patented Oct. 26, 1971 [73] Assignee Geigy Chemical Corporation Ardsley, N.Y.

[54] METHOD FOR THE DETERMINATION OF PSEUDOMONAS IN BIOLOGICAL MEDIA 8 Claims, No Drawings [52] U.S.Cl 195/1035,

195/100 [51] Int. Cl C12k 1/06 [50] Field of Search 195/1035, 100, 101, 102; 260/611 [56] References Cited UNITED STATES PATENTS 3,284,362 11/1966 Zussman 424/340 X 3,405,184 10/1968 Widiger et a1. 424/340 X Primary Examiner-A. Louis Monacell Assistant Examiner-Max D. Hensley Altorneys- Karl E .Iorda and Bruce M. Collins ABSTRACT: The presence of Pseudomonas in biological media containing other micro-organisms is determined by adding to the media from 100 to about 800 parts per million ofa compound of the formula:

HO R I wherein R is halogen and R is hydrogen or chloro.

The medium is then maintained at 20 to 40 C. for an incubation period of 12 hours to 14 days. A particularly preferred compound useful in this method is 2,4,4-trichloro-2'-hydroxydiphenyl ether METHOD FOR THE DETERMINATION OF PSEUDOMONAS IN BIOLOGICAL MEDIA BACKGROUND OF THE INVENTION Finding an effective means for the isolation and detection of bacteria belonging to the genus Pseudomonas has been long sought by those working in the biological and medical arts. A simple and rapid means of isolating and identifying these bacteria is highly desirable since they are responsible for numerous pathogenic disturbances in humans, animals and plants.

The charred skin of burn patients is particularly susceptible to Pseudomonas with resultant serious infections and sometimes death, Pseudomonas is often involved in urinary, intestinal and conjunctival infections.

Pseudomonads are responsible for numerous plant diseases, such as lettuce leaf rot; and for the deterioration of various materials such as food, paint, latex, plastics, asphaltic material and various liquids, creams and lotions employed for cosmetic and pharmaceutical purposes.

Up to the time of the present invention, there has been no rapid method available for isolating and differentiating these Pseudomonas from other micro-organisms present in clinical body fluids or on other substrates.

The usual procedure for isolating and differentiating Pseudomonads from other micro-organisms involves plating the samples onto a Gram-differentiating media, having a trained bacteriologist pick presumptive colonies from the media, and then subjecting the organisms to various biochemical tests. Under ideal conditions and in the hands of highly trained personnel, a positive diagnosis of Pseudomonas required 3 to 4 days. The direct quantitative enumeration of Pseudomonas in a sample containing a mixture of micro-organisms has been an almost impossible task.

This invention provides a simple and efficient method of isolating and detecting Pseudomonas in varied biological media containing Pseudomonas and any other micro-organisms.

According to this invention, (a) a nutrient medium is provided which includes from about 100 to 800 parts per million of a compound of the formula OH R l wherein R is halogen and R, is hydrogen or chloro, (b) said medium is inoculated with a sample containing unknown micro-organisms, (c) said medium is maintained at a temperature of about to about 40 C. for an incubation period of 12 hours to 14 days, and (d) said medium is observed for the presence of Pseudomonas.

Additionally, methods are provided for the simultaneous isolation and differentiation of Pseudomonas strains producing fluorescent pigments, pyocyanin pigments or hydrogen sulfide.

A particularly preferred compound of the above formula for use in the method of this invention is 2,4,4-trichloro-2'- hydroxydiphenyl ether. It is stable to steam sterilization and does not interfere with other usual ingredients added to form a complete biological medium, for example, metal salts, buffers, blood, proteins, liquids, indicators and carbohydrates. The reagent is not pH sensitive, and generally any medium having a pH within the range of 3.0 to 9.0 is suitable for the practice of this invention.

While an addition of 100 parts per million of the 2,4,4- trichloro-2-hydroxydiphenyl ether is generally sufficient to differentiate between all of the micro-organisms and Pseudomonads, it is generally preferred to add between about 300 and about 500 parts per million to further insure that all other colonies except Pseudomonads have been eliminated. Up to about 800 parts per million may be added, but greater amounts are wasteful and approach the point where the growth ofstrains of Pseudomonads will be inhibited.

The following examples describing certain representative embodiments of this invention will serve to further illustrate the nature of the invention. lt is to be understood that the examples are merely illustrative and intended to enable those skilled in the art to practice the invention in all of the embodiments flowing therefrom and do not in any way limit the scope of the invention defined in the claims.

EXAMPLE I Various commercial biological media containing agar were dissolved in distilled water and sterilized for 10 to 15 minutes at 15 pounds pressure (121 C.). The media were cooled to 50 C. and varying amounts of 2,4,4'-trichloro-2'-hydroxydiphenyl ether added from a filter sterilized solution of the compound in methyl cellosolve. The media containing the various indicated levels of 2,4,4-trichloro-2'-hydroxydiphenyl ether were then inoculated with pure cultures of indicated micro-organisms and solidified in petri dishes. After a suitable period of incubation l-lO days) the plates were inspected for visible signs of growth.

As indicated in the following table I, all of the wide representative spectrum of micro-organisms which included bacteria, fungi and yeast which were tested failed to grow when 2,4,4'-trichloro-2-hydroxydiphenyl ether was present in an amount of parts per million or more. The Pseudomonas, however, fluorished in all media which contained as high as 300 parts per million of 2,4,4'-trichloro-2'-hydroxydiphenyl ether.

TABLE I Inhibitory Microorganism coucvntrntion I Achro'mobacter bulyrz'cum, Lvvin 100 Achromobacter lipolylicimi, Levin 100 Aerobacter sp l0 icrostalagmus m in 10 Alcaligenes 31).. 1O Allemaria tennis 30 Aspcrgillus oryzae, ATCC 1011K 10H Aapergillus elm-alas" 30 Aspergillus fischeri. 10 Aspergillus flaws 30 Aspergillus 11 iger, (QM-458. 100 Bacillus mesentcricus, 3 Bacillus mycoides. 3 Bacillus pcrfringenm. 1U Bacillus sporogenes 3U Bacillus subtilzs l Brezribacterium ammonz'agenes:

ATCC6871 3 ATCC 6872- 3 Candida albicans. lll Chaclomium globosum l0!) Clostridium bulyricmn 30 Corynebaclerium acnes, ATM UJltl. l Coryncbaclerium diphtheroides:

10 100 lT. l0 Corynebactcrzum mmulmszmmn, 67-23. 1i) Epideromphyion floc0cs1m1 1U Escherichia cali:

ATCC4157 3 ATCC 4352. 3 C1 3 C2. i 3 C3- 3 NCTC 8106. 3 Fusarium ozyspurum l 100 Herrellea caginicola. l0 Keratinomyces ajelloi l0 Metarrhizium glulinosum 100 Micrococus 81), A20 Levin 0. 1 Micrococcus 3 A45 LtVil 0. l Microsporum cams 50 Mima polymorphm. 10 Manila nigra 30 Myrothecium 'z'errucarz 1U Paecilomg ces rarioli. l0 Penicillium citn'um ll) Penicillium chrysageneum 50 Penicillium italicum 10 Pery'g'llium notatum 50 Proteus mirabz'lis:

A'ICC E1921 1 ATCC 7002" 1 ATCC 4675 1 TABLE I Inhibitory Microorganism concentration 1 Proteus morga'nii, A'ICC 9237 3 Proteus rettgerz', ATCC 14505. 3 Proteus am'maniae, ATCC 4630- 3 Proteus zmlgarz's:

C 8427. 1 AT C 13315 1 ATCC 9920- 1 1 1 1 1 300 A1 300 A2. 300 A3 300 Pscudomonas fluorescens. 300 Paeudomonas fluorescens, Levin. 300 Pseu-domonas fragi 300 Pseudomonas pulrefaciem, Levin. 300 Pseudomo'nas pyocyanea 300 Rhizopus nigricans 10 Sacchoromg ces cereuisiae 10 Salmonella choleraesuis, A'ICC 10704.. 1 Salmonella cabana, ATCC 12007.." 1 Salmonella analum, AICC 9270. 1 Salmonella paratyphz' 3 Salmonella pullorum 1 Salmonella typhimuriam, ATCC 13311. 1 Salmonella typhosa, ATCC 6539.... 1 Salmonella shotmuelleri.... 1 Sarcina urea 3 Scopulariopsis brevicaulz's. 10 Shz'gella dysenteriae-. 3 Stachybotrys atia- 100 Staphylococcus albas 0. 1 Siaphylococcus aureus:

63 1 Staphylococcus epidermidis 10 Staphylococcus pyogenes (var. aureus): 1

S1 S2 1 S3 1 S4 1 S5. 1 $6-. 1 S7- 1 st ir ap y ococcus saprop ytz'cu Stemphyliam botryosumv. 30 Streptococcus j'aecalis. 1 Torula utilis 30 Trichoderma nirz'de 100 Trichophylon gypseum 10 Trichophyto'n interdigz'tale, ATCC 640. 10 Trz'chophyton rubru'm 10 1 In parts by million of 2,4,4-trichloro-2'-hydroxydiphenyl ether.

The following examples further demonstrate the effectiveness of 2,4,4-trichloro-2-hydroxydiphenyl ether in differentiating Pseudomonas from other micro-organisms. Typical media which may be prepared beforehand and have incorporated therein a suitable amount of 2,4,4'-trichloro-2- hydroxydiphenyl ether for use in the practice of this invention are illustrated.

EXAMPLE II A general purpose medium suitable for the isolation and differentiation of Pseudomonas species is prepared as follows:

Peptone 20 Magnesium chloride 1.4 g. Potassium sulfate 10.0 g. Agar [5.0 g. 2,4.4 Triehloro-2'- Hydmxydiphenyl Ether 0.25 g.

To rehydrate the medium, 46.65 g. of the above formulation is dissolved in distilled water and sterilized by autoclaving for minutes at 15 pounds pressure (121 C.). For an especially clear medium l0 grams of methyl cellosolve can be added to 1,000 ml. of the hot suspension prior to autoclaving. Alternatively, the methyl cellosolve can be added to the dry ingradients at the time of manufacture in which case the 2,4,4-

trichloro-2'-hydroxydiphenyl ether is first dissolved in the methyl cellosolve and this uniformly blended into the other portion of the formulation. As a second alternative, the formulation without the 2,4,4'-trichloro-2-hydroxydiphenyl ether can be compounded, rehydrated, sterilized, and the 2,4,4- trichloro-Z-hydroxydiphenyl ether added from a filter sterilized solution in methyl cellosolve.

As indicated in the following table ll, various strains of Pseudomonas grew on the above medium after 24 hours incubation at 37 C. while other bacteria normally growing in the above medium without 2,4,4-trichloro-2'-hydroxydiphenyl ether failed to grow.

TABLE II Micro-organisms Growth After 24 Hours at 37 C.

Pseudomonas aeruginora Growth Pseudamunarfluorescens Growth Fseudomona: pyocyanea G rowth Staphylococcus aureur No Growth Escherichia coli No Growth Proreur vulgaris No Growth Bacillus .rubtilis N 0 Growth Salmonella lyplrara No Growth EXAMPLE lll A medium particularly well suited for the simultaneous isolation and differentiation of Pseudomonas strains producing fluorescin pigments is prepared by incorporating 2,4,4- trichloro-Z-hydroxydiphenyl ether into the following ingredients by the methods described in example 11:

Proteose Peptone No. 3' 20 g.

Maltose 10 g. Dipotassium phosphate 1.5 Magnesium sulfate 0.3 g. 2,4 4'-trichloro-2'- hydroxydiphenyl ether 0.25 g. Agar 15 g.

Manufactured by Difeo Laboratories To rehydrate the medium, 47.05 g. of the powder is dissolved in 1,000 ml. of water and sterilized by autoclaving. As in example ll, methyl eellosolve is added at the time of manufacture, added later by the user prior to autoclaving or the base ingredients without 2,4,4'-trichloro-2'-hydroxydiphenyl ether prepared and the 2,4,4-trichloro-2'-hydroxydiphenyl ether added from a filter sterilized methyl cellosolve solution after rehydration and autoclaving.

While the base medium without 2,4,4'-trichloro-2'-hydroxydiphenyl ether can differentiate fluorescin producing strains of Pseudomonas previously isolated from other bacteria, the addition of 2,4,4'-trichloro-2'-hydroxydiphenyl ether renders isolation and differentiation possible in one step. Pseudomonas strains producing fluorescin are visualized on the above medium as yellow colonies; the pigment also dissipates into the surrounding agar. The production of pyocyanin pigments associated with certain other strains of Pseudomonas is generally inhibited.

EXAMPLE IV A medium particularly well suited for the simultaneous isolation and differentiation of Pseudomonas strains producing pyocyanin pigments is prepared by incorporating 2,4,4-

trichloro-2'-hydroxydiphenyl ether into the following ingredients by the methods described in examples ll and Ill.

Peptone 20 g. DL-Alanine 2 g, Sodium citrate l g. Potassium sulfate 86 g. Potassium chloride 1.4 g. Magnesium sulfate 2,4,4'-trichloro-2- hydroxydiphcnyl ether 0.25 g. Agar l g.

The medium is rehydrated by dissolving 58.65 grams of the medium in 1,000 ml. of distilled water and sterilizing by autoclaving. As in examples ll and Ill methyl cellosolve can be added at the time of manufacture, added later by the user prior to autoclaving or the base ingredients without 2,4,4- trichloro-Z-hydroxydiphenyl ether prepared and the 2,4,4- trichloro-2-hydroxydiphenyl ether added from a filter sterilized methyl cellosolve solution after rehydration and autoclavmg.

Strains of Pseudomonas producing pyocyanin produce blue colonies; the pigment also diffuses into the surrounding agar. The production offluorescin pigment is generally inhibited.

EXAMPLE V A medium well suited to isolate and simultaneously differentiate Pseudomonas strains producing hydrogen sulfide is prepared by incorporating 2,4,4'-trichloro-2'-hydroxydiphenyl ether in the following formulation:

Peptone [5 g. Proleose Peptone 5 g. Ferric Ammonium Citrate 0.5 g. Dipotassium phosphate 1 g. Sodium thiosulfatc 0.08 g. Agar l5 g. 2,4,4'-Trichloro-2- hydroxydiphenyl ether 0.25 g

The medium is rehydrated by dissolving 58.65 grams of the medium in 1,000 ml. of distilled water and sterilizing by autoclaving. Alternatively the 2,4,4'-trichloro-2-hydroxydiphenyl ether is added as described in examples ll through lV.

Cultures of Pseudomonas putrefaciens are isolated as black colonies while other strains of Pseudomonas are grayish-tan.

Although the present invention has been described with preferred and specific embodiments, it is to be understood that modifications and variations may be resorted to, without departing from the spirit and scope of this invention, as those skilled in the art will readily understand. Such variations and modifications are considered to be within the purview and scope of the appended claims.

What is claimed is:

1. A method for the detection and isolation of Pseudomonas which comprises a. providing a nutrient medium which includes from about 100 to about 800 parts per million of a compound of the formula wherein R is halogen and R is hydrogen or chloro,

b. inoculating said medium with a sample containing unknown micro-organisms, c. maintaining said medium at a temperature of about to about 40 C. for an incubation period of 12 hours to 14 days, and

d. observing said medium for the presence of Pseudomonas.

2. A method as claimed in claim 1 wherein from about 300 to about 500 parts per million of 2,4,4'-trichloro-2-hydroxydiphenyl ether is present in said nutrient medium.

3. A method for the simultaneous isolation and differentiation of Pseudomonas strains producing fiuorescin pigments which comprises a. providing a nutrient medium comprising proteose peptone, maltose, dipotassium phosphate, magnesium sulfate, agar and from about to about 800 parts per million ofa compound of the formula wherein R is halogen and R, is hydrogen or chloro,

b, inoculating said medium with a sample containing unknown micro-organisms,

c. maintaining said medium at a temperature of about 20 to about 40 C. for an incubation period for 12 hours to l4 days, and

d. observing said medium for the presence of said Pseudomonas strains as yellow colonies.

4. A method as claimed in claim 3 in which from about 300 to about 500 parts per million of 2,4,4'-trichloro-2'-hydroxydiphenyl ether is present in said nutrient medium.

5. A method for the simultaneous isolation and differentiation of Pseudomonas strains producing pyocyanin pigments which comprises a. providing a nutrient medium comprising peptone, DL-

alanine, sodium citrate, potassium sulfate, agar and from about 100 to about 800 parts per million of a compound of the formula wherein R is halogen and R is hydrogen or chloro, b. inoculating said medium with a sample containing unknown micro-organisms, c. maintaining said medium at a temperature of about 20 to about 40 C. for an incubation period of 12 hours to l4 I wherein R is halogen and R is hydrogen or chloro,

domonas strains as blacli eolonies.

8. A method as claimed in claim 7 in which from about 300 to about 500 parts per million of 2,4,4'-trichloro2'-hydroxydiphenyl ether is present in said nutrient medium.

l i i I i 

2. A method as claimed in claim 1 wherein from about 300 to about 500 parts per million of 2,4,4''-trichloro-2''-hydroxydiphenyl ether is present in said nutrient medium.
 3. A method for the simultaneous isolation and differentiation of Pseudomonas strains producing fluorescin pigments which comprises a. providing a nutrient medium comprising proteose peptone, maltose, dipotassium phosphate, magnesium sulfate, agar and from about 100 to about 800 parts per million of a compound of the formula
 4. A method as claimed in claim 3 in which from about 300 to about 500 parts per million of 2,4,4''-trichloro-2''-hydroxydiphenyl ether is present in said nutrient medium.
 5. A method for the simultaneous isolation and differentiation of Pseudomonas strains producing pyocyanin pigments which comprises a. providing a nutrient medium comprising peptone, DL-alanine, sodium citrate, potassium sulfate, agar and from about 100 to about 800 parts per million of a compound of the formula
 6. A method as claimed in claim 5 in which from about 300 to about 500 parts per million of 2,4,4''-trichloro-2''-hydroxydiphenyl ether is present in said nutrient medium.
 7. A method for the simultaneous isolation and differentiation of Pseudomonas strains producing hydrogen sulfide which comprises a. providing a nutrient medium comprising peptone, proteose peptone, ferric ammonium citrate, dipotassium phosphate, sodium thiosulfate, agar and from about 100 to about 800 parts per million of a compound of the formula
 8. A method as claimed in claim 7 in which from about 300 to about 500 parts per million of 2,4,4''-trichloro-2''-hydroxydiphenyl ether is present in said nutrient medium. 